I. Cloning of a cellular thyroid hormone binding protein (p55): A cellular thyroid hormone binding protein was previously purified to homogeneity. This protein was found concentrated on the lumenal face of the endoplasmic reticulum and nuclear envelope. A cDNA encoding p55 was cloned and its sequence was determined. It contains a single open reading frame of 1524 nucleotides which encodes a polypeptide of 491 amino acids and a putative signal sequence of 17 amino acids. The deduced amino acid sequence shows excellent correspondence with the two partial protein sequences. In vitro translation of mRNA prepared by transcription of the cDNA clone with T7 RNA polymerase after cloning into pGEM3 yielded proteins which were immunoprecipitated by monoclonal and polyclonal antibodies against p55. The isolation of the cDNA clone should allow elucidation of the cellular function of p55. II. Regulation of p55 by 3,3', 5-triiodo-L-thyronine (T3): The effect of T3 on the stability and synthesis of p55 was evaluated. Rat pituitary tumor GH3 cells were grown in regular, thyroid hormone-depleted (Td) and Td supplemented with T3 medium. Immunoprecipitation of 35S-methionine-labeled cellular extracts indicated that p55 is two-fold more abundant in cells grown in Td medium than in cells in regular or Td + T3 medium. Northern blot analysis indicated that no difference in mRNA level was detected in cells grown in three different conditions. Pulse-chase experiment indicated that p55 is two-fold more stable in cells grown in Td medium. Thus the down regulation of p55 by T3 occurs at a post-translational level. III. Purification and characterization of another thyroid hormone binding protein (p58): Analysis of the cellular extracts of A431 cells indicated the presence of another binding protein for T3. In contrast to p55, this binding component has an apparent MW of 58K. p58 has been purified to homogeneity. Antibodies to p58 are being prepared and its cellular functions will be evaluated.